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Molecular characterization of the plasma membrane H(+)-ATPase, an antifungal target in Cryptococcus neoformans
P Soteropoulos1, T Vaz, R Santangelo
1Public Health Research Institute, New York, NY 10016, USA.
Abstract:
The Cryptococcus neoformans PMA1 gene, encoding a plasma membrane H(+)-ATPase, was isolated from a genomic DNA library of serotype A strain ATCC 6352. An open reading frame of 3,380 nucleotides contains six introns and encodes a predicted protein consisting of 998 amino acids with a molecular mass of approximately 108 kDa. Plasma membranes were isolated, and the H(+)-ATPase was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be slightly larger than the S. cerevisiae H(+)-ATPase, consistent with its predicted molecular mass. The plasma membrane-bound enzyme exhibited a pH 6.5 optimum for ATP hydrolysis, K(m) and V(max) values of 0.5 mM and 3.1 micromol mg(-1) min(-1), respectively, and an apparent K(i) for vanadate inhibition of 1.6 microM. ATP hydrolysis in plasma membranes and medium acidification by whole cells were inhibited by ebselen, a nonspecific H(+)-ATPase antagonist which was also fungicidal. The predicted C. neoformans protein is 35% identical to proton pumps of both pathogenic and nonpathogenic fungi but exhibits more than 50% identity to PMA1 genes from plants. Collectively, this study provides the basis for establishing the Cryptococcus H(+)-ATPase as a viable target for antifungal drug discovery.
Insights
Researchers identified the Cryptococcus neoformans PMA1 gene, encoding a crucial plasma membrane H(+)-ATPase. This enzyme is a potential new target for developing antifungal drugs.
Area of Science:
- Mycology
- Molecular Biology
- Biochemistry
Background:
- Cryptococcus neoformans is an opportunistic fungal pathogen.
- Plasma membrane H(+)-ATPases are essential for fungal cell homeostasis.
- Identifying novel antifungal targets is critical for combating resistant strains.
Purpose of the Study:
- To isolate and characterize the PMA1 gene and its encoded H(+)-ATPase in Cryptococcus neoformans.
- To evaluate the potential of Cryptococcus H(+)-ATPase as a target for antifungal drug discovery.
Main Methods:
- Isolation of the PMA1 gene from a genomic DNA library.
- Expression analysis and protein characterization of the H(+)-ATPase.
- Enzymatic assays to determine kinetic parameters and inhibition by vanadate and ebselen.
- Bioinformatic analysis of protein sequence identity.
Main Results:
- The PMA1 gene encodes a 998-amino acid protein (108 kDa) with six introns.
- The purified H(+)-ATPase showed optimal activity at pH 6.5 with specific kinetic parameters (K(m)=0.5 mM, V(max)=3.1 micromol mg(-1) min(-1)).
- Vanadate inhibited the enzyme (K(i)=1.6 microM), and ebselen inhibited ATP hydrolysis and cell acidification, exhibiting fungicidal activity.
- The C. neoformans H(+)-ATPase shares 35% identity with fungal homologs and over 50% with plant PMA1 genes.
Conclusions:
- The Cryptococcus neoformans PMA1 gene and its protein product, H(+)-ATPase, have been successfully isolated and characterized.
- The enzyme's kinetic properties and sensitivity to inhibitors suggest it is a viable target for antifungal drug development.
- Further research into this H(+)-ATPase could lead to novel therapeutic strategies against Cryptococcus infections.
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